Membrane proteins (MPs) comprise a third of proteins encoded in genomes and perform essential functions as receptors, transporters and channels. However, their isolation and purification is challenging due to their hydrophobic nature. Conventionally, detergents are used to solubilize MPs from their native lipid environment and stabilize them in aqueous buffer(1). However, most detergents display complicated phase behavior depending on concentration, ionic strength and the presence of other lipids and proteins. Successful methods to reconstitute membrane proteins thus rely on the unique behavior of detergents. Driven by the importance of membrane protein studies, a variety of amphiphilic reagents, including amphiphilic polymers(2), protein-based nanodiscs(3), peptide-based detergents(4), maltose neopentyl glycol detergents(5), etc., have been developed over the past decades to facilitate functional and structural studies of membrane proteins.
In an earlier work, Tao and colleagues reported the engineering of three β-sheet peptides (BPs), namely BP1, BP2 and BP3, to stabilize membrane proteins(6). The Tao reference is incorporated herein by reference in its entirety. These β-sheet peptides are 8-amino-acid peptides with alternating polar and apolar residues and an octyl side chain at each end. The β-sheet peptides of Tao et al. share the core structure acetykoctyl)Gly-Ser-Leu-Ser-Leu-Asp-(octyl)Gly-Asp-NH2 (SEQ ID NO:1) with differing numbers of N-methyl amino acids, as shown in structure (1):

The N-methyl amino acids differ between the three constructs, BP1, BP2 and BP3 disclosed by Tao et al. as follows: for BP1 (referred to as structure (2)), R1=R3=H, R2=Me. For BP2 (referred to as structure (3)), R1=R2=Me, R3=H. For BP3 (referred to as structure (4)), R1=R2=R3=Me.
Because of the special sequence design, these β-sheet peptides self-assemble into filaments in solution and restructure into a β-barrel upon association with membrane proteins (MPs). The peptides are eight amino acids in length to span the hydrophobic region of a lipid bilayer (a distance of approximately 3 nm), and, without being bound by theory, are believed to sequester the hydrophobic surfaces of the membrane protein by forming an ordered, stabilizing β-barrel-like structure. The resulting BP:MP complexes prevented membrane proteins from aggregation when diluted in detergent-free buffer. Due to the inter-strand hydrogen bond interaction, β-sheet peptides are much less likely to dissociate from the membrane proteins once the BP:MP complex has formed.
Aquaporins are a group of integral membrane proteins that conduct water through the cellular membrane with exceptional selectivity and permeability. More specifically, aquaporins selectively conduct water molecules in and out of cells, while preventing the passage of ions and other solutes. Water molecules traverse through the pore of the channel in single file. The presence of water channels increases membrane permeability to water, while the pores are impermeable to charged species, which helps to preserve a membrane's electrochemical potential difference relative to the surrounding environment.
As illustrated in FIG. 1A, aquaporins use a combination of two different mechanisms to ensure that only water molecules pass through the protein. First, the aquaporin has an hourglass-shaped channel that narrows at its middle. The narrow size of the channel provides a size restriction to help control the size of molecule that can pass through the channel. Second, the presence of a positive charge within the channel helps to prevent protons from passing through the channel. Aquaporins are made up of six transmembrane α-helices arranged in a right-handed bundle. There are five interhelical loop regions (A-E). Loops B and E are hydrophobic loops that contain a highly conserved asparagine-proline-alanine (NPA) motif, which overlaps the middle of the lipid bilayer of the membrane, forming a three-dimensional hourglass structure where water flows through the pore. Aquaporins form tetramers in the cell membrane, with each monomer acting as a water channel. Water can flow in either direction through the pores of the aquaporin, for example due to hydraulic or osmotic pressure.
Aquaporins are highly conserved. See, for example, Erbakan, M., et al. (2014). The sequence alignment carried out in this paper, shown in FIG. 1B, shows the high degree of conservation between aquaporins in diverse species including Rhodobacter sphaeroides ATCC 17023 (RsAqpZ, ABA78939.1, SEQ ID NO:2), Escherichia coli K12 (EcAqpZ, BAA08441.1, SEQ ID NO:3 and GlpF, AFH13815.1, SEQ ID NO:4), Methanobacter marburgensis (AqpM, ADL58146.1, SEQ ID NO:5), Synecoccocus elongatus (SsAqpZ, AAM82672.1 SEQ ID NO:6), Pichia pastoris (AQY1, CCA39392.1, SEQ ID NO:7), Spinacia oleracea (SOPIP2; 1, AAA99274.2, SEQ ID NO:8) and Homo sapiens (AQP1, NP 932766.1, SEQ ID NO:9; AQP4, AAH22286.1 SEQ ID NO:10; AQP9, NP 066190.2, SEQ ID NO:11).
Many members of the aquaporin family, for example, aquaporin Z (AqpZ) from Escherichia coli, are very rugged and can withstand harsh conditions without losing their function. For example, AqpZ resist denaturation from exposure to acids, voltages, detergents and heat.
Aquaporins are naturally embedded in a hydrophobic lipid bilayer environment and are normally unstable proteins that cannot function as free molecules outside of a membrane. Previous attempts have been made to use aquaporin to purify water. For example, a Langmuir-Blodgett film was devised which attempted to use aquaporin in thin monolayers to purify water, but such constructs were unstable and difficult to scale (see e.g. Sun et al., 2012). To increase stability, monolayers were tethered to support substrates and a polymer cushion was used to dampen vibration (see e.g. Sun et al., 2013); however, these constructs also proved to be unstable and difficult to produce.
The foregoing examples of the related art and limitations related thereto are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.